Tuberculosis causes more deaths annually world-wide than any other infectious human pathogen. Eradication of tuberculosis will require development of an effective vaccine, which in turn hinges on understanding the mechanism of protective immunity against tuberculosis. Immune responses to M. tuberculosis are thought to be mediated by T lymphocytes that recognize mycobacterial antigens. Antigen recognition is mediated by T cell receptors composed of alpha and beta chains, or gamma and delta chains. Although the functions of gammadelta T cells remain unknown, there is growing evidence that they are involved in the immune response to mycobacteria and other pathogenic microorganisms. The gaol of this proposal is to investigate the role of lymphocytes bearing gammadelta T cell receptors in mediating resistant and protective immune responses to Mycobacterium tuberculosis. Our specific aims are: 1) Determination of the distribution of gammadelta T cells and their subpopulations in tissue and blood of patients throughout the spectrum of tuberculous infection, using single and double immunostaining. Antigen reactivity of gammadelta T cells will be evaluated by measurement of the percentage of gammadelta T cells after culture in vitro with M. tuberculosis. 2) Characterization of mycobacterial antigens and epitopes recognized by gammadelta T cells by establishing gammadelta T cell lines and clones from patients with tuberculous infection and testing their reactivity to mycobacterial antigens directly or by the T-cell Western blot technique. Epitope definition will be achieved by testing gammadelta T cell clones against synthetic overlapping peptides. 3) Evaluation of the functional capacity of gammadelta T cells by determining their pattern of lymphokine secretion and cytotoxic capacity when triggered by mycobacterial antigens. 4) Determination of the degree of diversity of the genes coding for the gamma and delta chains of the T cell receptor throughout the spectrum of tuberculous infection and at the site of disease activity, using PCR to amplify relevant gene segments. Limited diversity would suggest that gammadelta T cells recognize highly conserved antigens such as heat shock proteins. The studies proposed will provide information on fundamental questions regarding the function of gammadelta T lymphocytes in the immune response to infectious pathogens.